Abstract
This chapter covers topics including: (1) timing of sperm retrieval, particularly whether or not it should be performed in conjunction with oocyte retrieval from the female partner; (2) processing of surgically retrieved sperm for intracytoplasmic sperm injection (ICSI) –which are the most appropriate types of sperm preparation techniques in cases of epidydimal- or testicular-retrieved spermatozoa, and low or high numbers of retrieved spermatozoa; (3) selection of surgically retrieved sperm for ICSI, including methods for the selection of immotile spermatozoa, if needed, such as mechanical touch technique, hypoosmotic swelling test, chemical motility enhancers (e.g., pentoxifylline and theophylline), laser-assisted immotile sperm selection, and birefringence–polarization microscopy; (4) artificial oocyte activation, which may be useful in those selected patients who might have low fertilization potential, as well as the potential benefits of mechanical activation, electrical stimulation, and chemical activation on ICSI outcomes.
11.1 Introduction
In 1992, a major development in the field of male infertility and assisted reproduction, intracytoplasmic sperm injection (ICSI), was introduced. However, it was only a few years later that the successful use of sperm retrieved directly from epididymis and testes was reported for the treatment of male infertility by virtue of azoospermia.
Azoospermia, observed in 1 percent of the general population and in 10–15 percent of infertile men, is the absence of sperm in the ejaculate after assessment of at least two centrifuged semen samples, and is clinically categorized as obstructive azoospermia (OA) or nonobstructive azoospermia (NOA) [1].
Successful sperm retrieval is possible in practically 100 percent of patients with OA and in 50 percent of patients with NOA using the following techniques that capture spermatozoa directly from the epididymis and testes: percutaneous epididymal sperm aspiration (PESA), microsurgical epididymal sperm aspiration (MESA), testicular sperm aspiration (TESA), testicular sperm extraction (TESE), and microdissection testicular sperm extraction (micro-TESE) [2]. Despite the fertilization rate being higher with epididymal sperm, similar clinical pregnancy and implantation rates are obtained with testicular or epididymal spermatozoa, regardless of whether the azoospermia is obstructive or nonobstructive [3]. Also, there is evidence that the success of the first TESA procedure predicts the results of additional attempts in NOA patients [4].
This chapter summarizes the development of the processing and selection of surgically retrieved sperm, as well as the use of oocyte activation in couples with azoospermia.
11.2 Timing of Sperm Retrieval
Sperm harvesting can be performed prior to or in conjunction with oocyte retrieval from the female partner. There are advantages and disadvantages associated with both method schedules; ultimately, the decision is made according to the assisted fertilization center’s preference.
Despite the difficulties in scheduling sperm retrieval in conjunction with oocyte retrieval, most of the centers prefer to handle fresh sperm samples rather than frozen. Thus, sperm harvesting is performed the day of egg retrieval, mainly in male partners with known OA, in which sperm retrieval is supposed to be a simple procedure.
Nevertheless, harvesting sperm prior to oocyte retrieval obviates several concerns related to the center’s availability of operating and recovery rooms, surgeons, and embryologists in the busy morning hours. Moreover, it has to be considered that, in many cases, the oocyte retrieval day is not on the date originally scheduled. Both epididymal and testicular sperm are able to survive for up to 72 hours if properly incubated in culture media. In fact, sperm extracted directly from the tubules of the testicular tissue may take a while to become motile. In addition, sperm motility can be induced with a motility enhancer for the selection of viable sperm for ICSI.
When the odds of finding sperm are low, there is always the option of harvesting and freezing sperm in advance. If no sperm is retrieved, the couple has to choose between using donor sperm or freezing the entire cohort of retrieved oocytes until another sperm retrieval technique can be scheduled. Similar ICSI outcomes are yielded when freeze–thawed or fresh, testicular or epididymal spermatozoa are used, particularly when high numbers of spermatozoa are harvested [5].
Whenever micro-TESE is indicated, it is usually performed the day before oocyte retrieval due to the complexity of the procedure [6]. It is important to mention that some couples may be reluctant to undergo surgery together because of personal reasons, such as lack of home assistance and transportation, among others.
11.3 Processing of Surgically Retrieved Sperm for ICSI
Epididymal aspirates, such as MESA and PESA, are generally mixed with buffered culture medium before an aliquot is used for count, motility, and morphology analysis. Aspirates containing low sperm count can be processed by double washing and centrifugation, while those with high sperm count can be treated with density gradient centrifugation followed by double washing [reviewed in 7]. An aliquot of the processed sample is placed on a Petri dish and covered with oil until the moment of ICSI. Supernumerary sperm can be frozen and used in subsequent ICSI cycles.
Sperm aspirated directly from the testis are poured in a dry tube or in culture medium droplets under oil until sperm selection for ICSI. For sperm retrieved through TESE specimens, different processing has been described, such as mechanical processing, and the use of enzymes or erythrocyte-lysing buffer to improve sperm recovery. A method of mechanical testicular tissue homogenization using a loose-fitting glass pestle, followed by repeated aspiration through a 16 G needle, has been proposed by Oates et al. [8].
One study compared four mechanical methods to retrieve testicular spermatozoa – rough shredding, fine mincing, vortexing, and crushing in a grinder with pestle – and demonstrated that the most effective method regarding motile sperm count and morphology was fine mincing [9]. In fact, rupture of seminiferous tubules by shredding and fine mincing is the most used testicular tissue processing method. The testicular tissue is placed in a Petri dish containing buffered culture medium and shredded with needles, microscope slides, or scissors, thus releasing sperm into the medium. The sample is assessed under an inverted microscope or a phase-contrast microscope. Upon the easy identification of sperm, the sample is allowed to rest for the sedimentation of the remaining tissue pieces, and then the supernatant is centrifuged, removed, and resuspended. The sample is smeared on a Petri dish overlaid with oil, from which the sperm will be selected from ICSI.
Red blood cell contamination in the testicular tissue sample is a recurrent feature that may hinder the identification of spermatozoa. In those cases, the tissue sample may be exposed to an erythrocyte-lysing buffer (ELB) after shredding. This method improves sperm recovery and selection, and reduces the sperm identification interval [10].
In men presenting with limited sperm production, mechanical sperm extraction can be complicated and time-consuming, and thus testicular sperm can be extracted enzymatically to improve sperm recovery. The most common enzymes used to digest testicular tissue are type IA and IV collagenases. The collagenases are able to digest the collagen present in the membranes and extracellular matrix. It has been demonstrated that the use of collagenase results in the recovery of sperm in nearly 26 percent of men undergoing ICSI, after no sperm had been found post-mechanical shredding [10]. Therefore, enzymatic digestion is routinely used after failure of sperm identification with mechanical shredding. Even though the effectiveness of the enzymatic methods on testicular sperm recovery has already been proven, the mechanical approaches are more widespread and routinely applied.
There are still several challenges hindering the management of infertility in men with NOA. Clinically, there is a need to develop diagnostic tests for the prediction of successful testicular sperm retrieval. At the laboratory level, some methods have already been developed to enhance sperm selection in those patients.
11.4 Selection of Surgically Retrieved Sperm for ICSI
Successful retrieval of sperm is only the first step toward the achievement of successful ICSI outcomes. In fact, ICSI performed with ejaculated sperm already made us wonder about the potential impact of the non-natural sperm selection that is performed by the embryologist. This question is even more pressing when testicular sperm is used for ICSI. Therefore, methods to aid in the selection of not only the “best-looking,” but also the most functional sperm are in order.
Extended culture of surgically retrieved spermatozoa may allow sperm maturation and attainment of motility; thus, several centers opt to perform sperm retrieval on the day before oocyte collection, and incubate the sperm sample until ICSI is performed. These improvements have been demonstrated in men with OA and NOA. It is noteworthy that sperm improvements peaked within 48 hours of culture, and intervals higher than two days were associated with sperm aging, represented by higher DNA damage and chromosomal abnormalities [11].
Nevertheless, even after extended culture of surgically retrieved sperm, some samples fail to provide motile sperm for ICSI. In those cases, the use of adjunct sperm selection techniques is recommended [reviewed in 7].
11.4.1 Mechanical Touch Technique
The mechanical touch technique, initially described as the sperm tail flexibility test, has been applied for the identification of immotile sperm viability prior to ICSI. It relies on the fact that immotile vital and non-vital sperm differ in tail flexibility when touched with the microinjection pipette, in which vital sperm are flexible and non-vital sperm are rigid. In the presence of tail flexibility, the sperm head will not move along with the tail, and the tail will return to its original positioning. Conversely, the sperm head will move together with a rigid tail when touched with the pipette, and the tail will not recover initial positioning.
Studies have demonstrated similar rates of fertilization, pregnancy, and delivery when motile testicular sperm and immotile testicular sperm selected with this technique were used for ICSI [12]. The advantages of the mechanical touch technique are that it is performed in real time, and it is free of dyes and chemicals; thus, it allows the analyzed sperm to be used for ICSI. In contrast, inter- and intra-observer variability may occur as a result of the subjectivity of the test, and it may be time-consuming to touch sperm by sperm before injection.
11.4.2 Hypoosmotic Swelling Test
The hypoosmotic swelling test evaluates the functional integrity of the plasma membrane, demonstrated by the capacity of the sperm to react to hypoosmotic media. Live sperm cells presumably possess intact and functional membranes, and will present tail swelling or curling due to water influx when exposed to hypotonic media, while dead sperm with disintegrated membranes will not react [7].
11.4.3 Chemical Motility Enhancers
Pentoxifylline and theophylline are methylxanthine derivatives used for treating vascular disorders due to their hemorrheological property. They are also phosphodiesterase inhibitors that can increase the levels of cyclic adenosine monophosphate, which plays a role in sperm motility. Pentoxifylline was introduced into the clinical routine of IVF centers in 1988; it took a decade to demonstrate its effects on immotile testicular sperm. The addition of pentoxifylline to sperm retrieved from the epididymis or testis resulted in motility gain and successful ICSI outcomes. The effects of theophylline on sibling oocytes were later investigated and the enhancement of sperm motility was confirmed.
Despite the sperm being washed post-methylxanthine treatment, the toxicity of these chemical compounds has been questioned. Nevertheless, so far, there is no evidence of abnormalities in the offspring, and thus the addition of methylxanthines may significantly reduce the time for sperm identification and selection, with no apparent detriment to the outcomes of pregnancy [7,13].
11.4.4 Laser-Assisted Immotile Sperm Selection
Laser-assisted immotile sperm selection (LAISS) was introduced in 2004 by Aktan et al. [14] and discriminates between viable and nonviable (dead) immotile sperm by assessing tail curling, as detected in live sperm when hit by a single laser shot close to the end of the tail. In contrast, the lack of tail dislocation demonstrates that the sperm is dead. This is a safe, chemical-free, and rapid sperm selection technique that provides an immediate response regarding sperm viability. Recently, testicular sperm selected via this technique demonstrated increased fertilization potential [15]. The main obstacle to its widespread application is the high cost of the equipment.
11.4.5 Birefringence: Polarization Microscopy
Pioneered by Baccetti [16], the analysis of birefringence (double reflection) in sperm cells, as confirmed by transmission electron microscopy, is an indicator of structural normality. The presence of birefringence suggests an organized and compact structure that reflects normal sperm nuclei, acrosomes, and flagella, whereas non-vital sperm lack birefringence because of their diverse texture. Therefore, the use of polarization microscopy for sperm selection prior to ICSI was proposed based on the properties of birefringence that human spermatozoon naturally possesses. The sperm selection via sperm head birefringence was demonstrated to be of benefit for oligoasthenoteratozoospermic samples and for testicular-retrieved sperm [17]. Nevertheless, the scientific literature concerning the efficacy of sperm selection using polarization microscopy is controversial [reviewed in 18], and reliable cut-off values are still needed. In addition, as polarization microscopy is an expensive asset to the IVF laboratory, cheaper sperm selection methods are available, thus hindering more widespread application of this technique.
11.5 Artificial Oocyte Activation
Sperm and egg interaction involves a series of physiological and biochemical events, involving species recognition, adhesion, and then fusion between gametes. The ultimate step, oocyte activation, is the starting point of a developmental program leading to the formation of a new individual [19]. Oocyte activation involves a characteristic pattern of intracellular calcium (Ca2+) oscillations, which orchestrate a series of further key events, such as cortical granule material exocytosis, prevention of polyspermy, polar body extrusion, cytoskeletal rearrangements, resumption of meiosis, formation of pronuclei, initiation of the first mitotic division in the new zygote, recruitment of maternal mRNA, and regulation of gene expression [20].
The exact mechanism responsible for oocyte activation has been a matter of debate for decades. Accumulating evidence has suggested that a catalytic substance, present in the sperm head, initiated Ca2+ release in the ooplasm following gamete fusion and the PLCζ (1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta-1) has been indicated as the key sperm oocyte activation factor [21].
It has been postulated that fertile men present a significantly higher proportion of sperm exhibiting PLCζ than infertile men. Reduced levels, abnormal localization, reduced activity/expression or genetic mutations in PLCζ have been associated with oocyte activation deficiency and therefore ICSI failure [22], even in patients with normal sperm parameters [23].
It has been reported that up to 70 percent of unfertilized, metaphase II oocytes after ICSI contain a swollen sperm head, indicating that the oocyte may have been correctly injected but failed to become activated to complete the second meiotic division [24].
Intracytoplasmic sperm injection was introduced to overcome severe male infertility, and although the procedure results in an average fertilization rate of 70 percent, in rare cases fertilization fails due to the lack of oocyte activation. Total fertilization failure occurs in 2–3 percent of ICSI cycles.
Oocyte activation failure can be compensated by artificially increasing calcium in the oocyte, the so-called artificial oocyte activation (AOA). Protocols used for AOA can be classified based on whether the mechanism evoking the Ca2+ trigger that promotes fertilization is mechanical, electrical, or chemical. However, one should keep in mind that ICSI AOA is not beneficial for patients with a suspected oocyte-related activation deficiency. Artificial oocyte activation is still an experimental technique and should be carefully considered.
11.5.1 Mechanical Activation
Mechanical oocyte activation entails a modified ICSI technique, in which the microinjection pipette is advanced during the ICSI procedure and peripheral cytoplasm is aspirated. Subsequently, the aspirated cytoplasm and the spermatozoon are deposited into the center of the oocyte.
Tesarik et al. [25] reported an ICSI technique primarily based on the repeated dislocation of the central ooplasm to the periphery, which increases the intracellular concentration of free Ca2+ either by creating an influx of Ca2+ or by the release of stored Ca2+ from cell organelles. It was suggested that this mechanical oocyte activation may have an immediate clinical application in patients with repeated fertilization failures, after ICSI, suspected to be caused by insufficiency of PLCζ or by a defective oocyte response to this sperm factor [25]. This technique may represent an alternative to the use of Ca2+ ionophores. The possibility of using a simple modification of the standard ICSI micromanipulation technique instead of ionophores alleviates concerns about the possible harmful effects on human embryos.
Considering a possible negative effect of this rather vigorous injection technique on further preimplantation development, Ebner et al. [26] developed a modified ICSI technique based on the hypothetical accumulation of highly polarized mitochondria. The cytoplasm in the periphery of the oocyte is thought to be rich in mitochondria with high inner-membrane potential and high metabolic ATP activity. Therefore, this method aims to accumulate peripheral mitochondria and thus increase energy sources at the site of subsequent pronuclear formation [26]. The authors suggested that the modified ICSI possibly accumulates mitochondria with a higher inner mitochondrial membrane potential and may be a reliable and safe alternative to conventional ICSI leading to comparable rates of blastocyst formation, implantation, and clinical pregnancy. In particular, this technology was proven to be useful in cases of previous failure of fertilization in ICSI cycles [26].
11.5.2 Electrical Activation
An electrical field can generate micropores in the cell membrane of gametes to induce sufficient Ca2+ influx through the pores to activate cytoplasm through a Ca2+-dependent mechanism. In animal models, oocytes injected with secondary spermatocytes or spermatids were fertilized when stimulated by electroporation and developed into normal offspring when the resultant embryos were transferred to a recipient uterus [27].
Yanagida et al. were the first to use ICSI followed by electrical oocyte activation for human oocyte activation, which resulted in healthy twins for a couple with previously failed fertilization after ICSI [28]. This study was followed by others with different experimental designs and different situations (i.e., previous fertilization failure, severe oligoasthenozoospermia, or NOA with total teratozoospermia).
Mansour et al. [29] evaluated the electroactivation of oocytes after ICSI in 241 cycles with either severe oligoasthenoteratozoospermia or azoospermia. For this trial, sibling oocytes for each patient were randomly divided after ICSI into two groups: the study group (electroactivation) and the control group (without electroactivation). Electrical activation resulted in a significant improvement in the fertilization rate after ICSI.
Oocyte electrical activation was also assessed in infertile couples having a history of total fertilization failure in previous ICSI cycles. For this study, a significantly increased fertilization rate and high-quality embryos rate was noted [30].
Some studies evaluated the effectiveness of oocyte electrical activation in non-fertilized oocytes after ICSI (rescue oocytes activation). Traditionally oocyte electrical activation is performed, on average, 30 min after ICSI. For the rescue oocyte activation, oocytes showing no evidence of fertilization by 16–24 hours after ICSI are electroactivated.
Zhang et al. [31] demonstrated that electrical stimulation can “rescue” oocytes that fail to fertilize by 24 hours after ICSI and stimulate them to complete the second meiotic division, form pronuclei, and undergo early embryonic development. One hundred failed-to-fertilize oocytes after ICSI were randomly assigned by stratified allocation according to oocyte grading before ICSI. Fifty unfertilized oocytes were electroactivated and the remaining 50 unfertilized oocytes were treated in the same way but without electrical activation. The embryo formation rates in the electrically activated group were 80 percent compared to 16 percent in the control group, suggesting once again that failed-to-fertilize oocytes after ICSI seem to be able to resume embryonic development after electrical activation. However, the study failed to demonstrate whether such embryos are capable of implanting.
It has been demonstrated that electroactivation results in a rapid rise in Ca2+ inside the oocyte, which gradually decreases to the original level in about 300 s. The aforementioned studies suggested that the oocyte electrical activation soon after ICSI or in unfertilized oocytes may be a promising approach for the treatment of patients with the risk of fertilization failure or those with high fertilization failure rates in previous cycles. However, electrical oocyte activation has not yet been proven to be the most efficient and safe method for oocyte activation in humans. Moreover, there is insufficient evidence available from randomized controlled trials to judge the efficacy and safety of this method in couples undergoing assisted reproduction cycles, and long-term follow-up studies are needed to ensure safety.
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